The present invention relates to a process for the manufacture of multi-microchannel elements and the use thereof.
Miniaturization is the recent trend in analytical chemistry and life sciences. Similar to advances with integrated circuits in the computer industry, the area of biological and chemical analysis is also undergoing a miniaturization effort. A key benefit of miniaturization is the prospect of integration of all of the steps of an analytical process into a single device. With micron-scale forming, technologies for fluid flows and biosensors are now converging and the way is open for full integration of microfluidics with micro- and even nano-scale biosensors. The range of components that have been miniaturized and used as building blocks for fully-integrated microanalytical systems includes pumps, valves, filters, mixers and reactors, sensors, three-dimensional networks of channels and electronic control circuitry. This will create monolithic structures for combined sampling and measurement, inclusive of special situations where arrays are needed.
Miniaturization of biosensor technologies has intrinsic advantages for improving resolution time (speed of assay), reducing reagent use, and allowing for higher sample throughput. A fusion of micro- and nanotechnology with biology has great potential for the development of low-cost disposable chips for rapid molecular analysis that can be carried out with simple handheld devices. It has been shown that conducting chemical or biological reactions in ultra narrow microchannels or porous materials allows a significant reduction of transport limitations across the unstirred layer (Nemst diffusion layer). The channels manufactured to date have dimensions from 1 to 300 μm and flow speeds in the range up to cm/s. The reduction of physical size of flow cells to cross-sectional dimensions on the order of tens or hundreds of microns (microchannels) results in a large surface area to volume ratio and a decrease of the Nemst diffusion layer from 10 μm to 0.02 μm. In this case, the binding events that occur within the ultra small volume of the microchannels do so with much higher efficiency than in the flow macroscopic systems. Since the magnitude of the diffusionally limited current is inversely proportional to the thickness of the diffusion layer, the effects observed in microchannels may be explained by facilitated diffusion of analytes (antigen, antibody, conjugate, substrate, products of enzymatic reactions, etc.) from the bulk of the solution to the electrode surface. Chips are already being fabricated with picoliter-sized wells and 10-microliter-sized chambers for sample preparation and detection.
Microanalytical devices have found many applications, ranging from the life sciences industries for pharmaceuticals and biomedicine (drug design, delivery and detection, diagnostic devices) to industrial applications of combinatorial synthesis (such as rapid chemical analysis and high throughput screening). Other areas of applications for microdevices for the transport of liquids and gases include fuel cells and optical applications. Miniaturization of biodetectors into a single integrated “lab-on-a-chip” system possesses great potential for environmental monitoring and point-of-care testing, and food analysis, which includes high sensitivity, improved accuracy, lower power and sample consumption, disposability and automation. An emerging demand is to monitor and detect chemical and biological warfare agents in real-time. Fast analysis and on-chip integration of supporting electronic circuitry for signal analysis and remote control would enable sensing at a remote location. The integration of microfluidic transport, total automation and materials handling contributes to a major reduction in system retention and material transfer losses, which reduces sample size requirements and cost of assay. Methods for the parallel in vitro screening of chemical and/or biological compounds are extremely important in drug development, functional proteomics studies, and clinical diagnostics and are used for the parallel screening of families of relative proteins. Current technological approaches which attempt to address this need include cell-based screening systems and microfluidics-based screening systems. Electrochemical microchip systems are particularly useful for this purpose because they easily interface the chemical and biological molecules in solutions with solid-state microelectrodes and can be directly integrated with microelectronics and microfluidic systems to gain advantages in miniaturization, multiplexing, and automation.
Most approaches developed to date are based on the application of two dimensional microchip formats, wherein a suitable set of biological receptor elements (enzyme, antibody, DNA, protein, etc.) are immobilized on the surface of a planar microchip substrate. The diffusion-controlled rate of biospecific reactions can be significantly accelerated in microfluidic systems through the use of microchannels or porous substrates that provide a unique means to prepare a three-dimensional network suited for the immobilization of different biomolecules. Heterogeneous flow-injection bioassays based on microchannel or microporous technologies offer extremely accelerated binding kinetics. First of all, there is a high surface area to volume ratio in the micro channels. Second, the flowing stream actively brings the sample in contact with the solid-phase antibody. This factor results in a greatly enhanced the rate of biospecific interaction (enzyme-substrate, ligand-receptor, antigen-antibody, DNA-DNA, etc.). By engaging the third dimension through organized porous or microchannels in a rigid support material the surface to volume ratio is significantly enhanced.
However, miniaturization and reduction in the volume of the sample analyzed in a microanalytical device have created a problem for analysis because the sample is no longer representative of the bulk specimen. For example, a 1 μL sample containing an analyte at a concentration of 1 fmol/L contains˜6000 molecules. Further reduction in sample size to 1 nL leads to a sample containing only 6 molecules of analyte, which may be substantially less than the detection limit of the analytical method formatted into the microchip. Another complication for microanalytical devices is evaporation of microvolumes of sample or reagents from the microchip.
In two dimensional microchip formats the density of receptor spots is ultimately limited by either the dispensing mechanism or the amount of biological recognition material within each spot. This fact negatively impacts the dynamic range and lower detection limit of analysis. The sensitivity of electrochemical detection based on microelectrodes is typically substantially lower then conventional techniques.
Currently, a single microchannel with special inner geometry is individually wired and used in microchip technology. But so far there is a large uncertainty in the test results because of variations in the properties of individual microchannels, i.e., there is no reproducibility of test results.
One of the barriers towards achieving true miniaturized total analysis systems is clogging problems which are related to the size of the microchannels. Micromachined from silicon, glass, plastics, and ceramics, the components have channels with size between 10-20 μm. Bubbles cause significant problems in micro-fluidic system applications. A single bubble can clog a channel that is 100 μm wide or less. Particles cause some of the same problems as bubbles, blocking fluid flow and clogging valves. Particles can enter through the route as well as through the packaging around the injection area. The clogging problems seriously limit the utility of the microfluidic devices. For example, they cannot be used to capture target analyte from samples that contain cellular or large molecule contaminants because the contaminants clog the pores or microchannels of the microfluidic system. Therefore, most biochips are designed for a single use only. Also, many fractionation methods require filters that become clogged over time and contribute to the carryover of particles between tests.
Review of known porous materials (microporous glass, porous silicon, microporous nylon membrane, porous carbon, porous Al2O3 sol-gel matrix) shows that their common disadvantage is that such structure does not allow good repeatability and reliability of biosensors. The structure usually has a three dimensional porous space including labyrinth and dead-end areas. In addition, the pore surface is rough, the pores have irregular shapes and there is non-uniform distribution in the electrodes. Due to the presence of labyrinths and dead-end areas, target analyte and products of chemical and biochemical reactions can accumulate in the porous electrode. This fact significantly decreases signal/noise ratio, which negatively affects the sensitivity of the assay.
Other limitations are complexity, low production rate, and high cost of known methods for manufacturing microporous and microchannel materials with a desired structure. These drawbacks are raised due to the necessity of involving a considerable number of complex operations involved in the manufacturing process, including having to machine channels into multiple components that then have to be joined together.
An important component in flow-through microchip technology is a micromixer. Rapid mixing is essential in many of the microfluidic systems used in biochemistry analysis, drug delivery and sequencing or synthesis of nucleic acids. Biological process such as cell activation, enzyme reactions and protein folding often involve reactions that require mixing of reactants for initiation. Mixing is necessary in lab-on-a-chip platforms. It is well known that the Reynolds number is low in microfluidic channels, and the flow is laminar under normal conditions. Therefore, the mixing of binary or multi-component fluid streams can be difficult in a microchannel, because it relies on diffusion. In general, micromixers are categorized as active and passive micromixers. Active micromixers use the disturbance generated by an external field for the mixing process. Active mixers can be categorized by the types of external disturbance effects such as pressure, temperature, electrokinetics, magnetohydrodynamics and acoustics. However, with external fields and the corresponding integrated components, the structures of active micromixers are often complicated and require complex fabrication processes. Furthermore, external power sources are needed for the operation of active micromixers. Thus, the integration of active mixers in a microfluidic system is both challenging and expensive. In contrast, passive micromixers do not require external energy; the mixing process relies entirely on diffusion or chaotic advection. Due to the dominating laminar flow on the microscale, mixing in passive micromixers relies mainly on molecular diffusion and chaotic advection.
The microscale passive mixing is a challenge because small channel dimensions make it difficult to create turbulence. In the microchannels liquids lose the assist that turbulence gives to mixing. The reason is that in laminar flow fluid laminae slide over each other and there is no turbulence. Typically, liquid flow in microfluidic devices has very low Reynolds numbers and molecular diffusion is responsible for the mixing and requires a long time to accomplish thorough mixing. The channel walls exert a drag on the liquid, so that fluid at the center of the channel moves faster that at the edge and concentrated samples quickly become smeared. At the microfluidic level, two liquids traveling side-by-side through a narrow channel only become fully mixed after many centimeters (>50 cm).
Currently, the fabrication of micromixers is based on technologies of micro electromechanical systems. The basic substrate materials are silicon, glass, and polymers. The basic design is a long microchannel with two inlets to its geometry; these designs are called the T-mixer or Y-mixer. Since the basic T-mixer entirely depends on molecular diffusion, a long mixing channel is needed. A recent simple method to reduce the mixing path is to make a narrow mixing channel, realizing parallel lamination with multiple streams. This mixer type was successfully used in a practical analysis. However, the limitation is that the fabrication processes is often complicated and expensive; it requires a complex multi step procedure.
In view of the foregoing, it is an object of the present application to avoid the aforementioned drawbacks, and therefore to provide a method of producing a multi-microchannel, flow-through element that, among other features, significantly improves assay sensitivity and the reproducibility of results.